Project Summary Pancreatic cancer is the fourth leading cause of cancer-related deaths in the US. Surgical resection is the only potentially curative treatment; however, only 15%-20% of patients present with tumors that can be resected. Currently there is no consensus regarding standard of care in unresectable cases, and the use of radiation to treat such cases is controversial because of the possibility of severe gastrointestinal (GI) toxic effects caused by the proximity of the pancreatic head to the duodenum. These toxic effects are thought to be secondary to radiation-induced DNA damage and subsequent depletion of rapidly dividing cells in the intestinal epithelium. Protecting the intestine from the toxic effects of radiation may enable dose escalation that could achieve more effective local control of disease. Fasting has been studied in mice as a protector against the lethal toxic effects of high-dose chemotherapy. In the applicant's laboratory, use of this mouse model to address the mechanism of protection showed that 24-h fasting protected intestinal stem cells after the administration of a toxic dose of etoposide. Based on this finding, the hypothesis of this proposal is that fasting protects mice from GI toxic effects caused by a lethal dose of total abdominal irradiation and allows escalation of the radiation dose for efficient killing of pancreatic tumor cells. Three aims are proposed. In Aim 1, the population of small intestinal stem cell(s) protected from high dose radiation by fasting will be identified and examined for proliferation, apoptosis and DNA repair capacity. In addition, genetically engineered mouse models of pancreatic ductal adenocarcinoma will be used to determine if fasting- mediated intestinal protection allows dose escalation and effective treatment of mice to increase survival and overall animal health. Histopathological analysis of malignant tissues will allow assessment of tumor response to radiation therapy after pre-treatment fasting. Experiments proposed in Aim 2 will identify epigenetic changes induced by fasting in small intestinal stem cells as well as associated gene expression changes. ChIP-Seq will be used to identify gene-specific changes in both activating and repressive marks that occur after 24 hours of fasting. Gene expression data will be used to determine which changes mediate increased DNA damage response gene expression changes in these cells. In Aim 3 the hypothesis that HDAC inhibitors (HDACi) and beta hydroxybutryate (?OHB) can function as intestinal radioprotectors will be tested. Intestinal stem cell enriched 3D spheroid cultures will be used to screen ?OHB and a panel of HDACi for their ability to provide protection from high dose radiation. Agents shown to provide radioprotection will be further evaluated for their ability to protect against or enhance repair of DNA damage by performing comet assays as well as histological evaluation of DDR protein foci formation and resolution. In addition, ChIP Seq and RNASeq will be performed to determine if changes induced in vitro correlate with those identified in vivo in Aim 2. Finally, agents that are found to provide radioprotection in vitro will be tested in vivo. Identifying the mechanism by which fasting confers intestinal protection will allow targeting with drugs and translation of these discoveries into the clinic, potentially widening the treatment window of radiation therapy for abdominal malignancies, such as pancreatic cancer. The potential to treat pancreatic cancer with high doses of radiation will give clinicians a new tool for combatting this disease.